Nk1 fragment of hepatocyte growth factor/scatter factor (hgf/sf) and variants thereof,and their use

ABSTRACT

The present invention relates to variants of the NK1 fragment of the polypeptide growth factor HGF/SF which act as agonists of the MET receptor and their use. The agonists comprise at least one substitution at positions equivalent to 132, 134, 170 and 181 of full length HGF/SF and these substitutions provide a variant which shows scatter factor activity and induces DNA synthesis. In vivo the variants provide protection from liver damage in a model of acute liver failure.

FIELD OF THE INVENTION

[0001] The present invention relates to variants of the NK1 fragment ofthe polypeptide growth factor HGF/SF which act as agonists of the METreceptor, and to the use of NK1 and its variants in methods oftreatment.

BACKGROUND TO THE INVENTION

[0002] The polypeptide growth factor hepatocyte growth factor/scatterfactor (HGF/SF) (Gherardi et al., 1989; Miyazawa et al., 1989; Nakamuraet al., 1989; Stoker et al., 1987) and its receptor MET, the product ofthe c-MET protoncogene (Bottaro et al., 1991), play essential roles inthe development of epithelial organs such as the placenta and liver(Schmidt et al., 1995; Uehara et al., 1995) and in the migration ofmyogenic precursor cells (Bladt et al., 1995) and motor neurons (Catonet al., 2000; Ebens et al., 1996).

[0003] HGF/SF and MET are also involved in the spreading of a variety ofepithelial tumours as a result of MET chromosomal rearrangements (Yu etal., 2000), somatic and/or germline mutations in the MET kinase (Schmidtet al., 1997) or, more often, over expression in tumour cells of anunrearranged and unmutated MET gene (reviewed in Jeffers et al., 1996).

[0004] HGF/SF has a unique domain structure that resembles that of theblood proteinase precursor plasminogen and consists of six domains: anN-terminal (N) domain, homologous to plasminogen activation peptide,four copies of the kringle (K) domain and a catalytically inactiveserine proteinase domain (Donate et al., 1994). Two products ofalternative splicing of the primary HGF/SF transcript encode NK1, afragment containing the N and the first K domain, K1, (Cioce et al.,1996)., and NK2, a fragment containing the N, K1 and second kringle, K2,domains (Chan et al., 1991; Hartmann et al., 1992; Miyazawa et al.,1991). Both NK1 (Lokker and Godowski, 1993) and NK2 (Chan et al., 1991)were initially characterized as MET antagonists, although experiments intransgenic mice have subsequently indicated that NK1 behaves in vivo asa bona fide receptor agonist (Jakubczak et al., 1998).

[0005] There is an important difference in the mechanism of receptorbinding and activation by HGF/SF and NK1. HGF/SF is fully active incells lacking heparan sulphate, while NK1 is only active in cells thatdisplay heparan sulphate or in the presence of soluble heparin (Schwallet al., 1996). Thus NK1, but not HGF/SF, resembles FGF (Rapraeger etal., 1991; Yayon et al., 1991) in terms of a requirement for heparansulphate for receptor binding and/or activation.

[0006] Early domain deletion experiments indicated that the N domain isimportant for heparin binding (Mizuno et al., 1994) and site-directedmutagenesis identified residues in this domain essential for binding(Hartmann et al., 1998). Thus reverse-charge mutation of R73 and R76decreased the affinity of HGF/SF for heparin by more than 50 fold(Hartmann et al., 1998). A role for several other positively-chargedresidues, such as K58, K60 and K62, was suggested from the solutionstructure of the N domain, as these residues are clustered in closeproximity of R73 and R76 (Zhou et al., 1998), and recent NMR experimentshave provided experimental support for an involvement of K60, K62, R73,R76, R78 and several other residues in heparin binding to the N domain(Zhou et al., 1999).

[0007] Despite this progress, the mechanism through which heparin andheparan sulphate confer agonistic activity to NK1 remains incompletelyunderstood. NK1 crystallizes as a dimer in the absence of heparin(Chirgadze et al., 1999; Ultsch et al., 1998), and the features of thisdimer suggested that it could represent the biologically active form ofNK1 (Chirgadze et al., 1999). No experimental evidence, however,supports this interpretation as yet.

[0008] Sequence Listing

[0009] SEQ ID NO:1 represents amino acids 28 to 210 of the human HGF/SFprotein. Residues 32-206 of HGF/SF are the wild type NK1 fragment. Wehave used short N- and C-terminal extensions as a matter of experimentalconvenience to optimise expression in yeast.

[0010] SEQ ID NO:2 is the full length HGF/SF sequence, of which residues1-31 are the leader sequence and 32-206 the NK1 fragment.

DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 shows DNA synthesis assays using MK cells. The cells werecultured to confluence in keratinocyte serum-free medium and transferredin basal medium for 24 hours before incubation with ³H-thymidine andHGF/SF or NK1 proteins at the concentrations (mol/L) indicated in theFigure (x-axis). DNA synthesis was measured as TCA-insolubleradioactivity; the Y axis shows ³H-thymidine incorporation,cpm×10³/well. The HP11 mutant is inactive and the HP12 shows muchreduced activity compared to wt-NK1. In contrast the 1K1 mutant is moreactive than wt-NK1 and full length HGF/SF.

[0012]FIG. 2 shows the survival rates of Balb/c mice afteradministration of a lethal dose of N-acetyl-p-aminophenol followed bytreatment with NK1 and a peptide of the invention.

[0013]FIG. 3 shows the survival rates of Balb/c mice afteradministration of a lethal dose of alpha-amanitin.

DISCLOSURE OF THE INVENTION

[0014] We have determined two X-ray crystal structures of NK1-heparincomplexes that define the heparin-binding site of NK1. Our analysis ofthese structures confirms that contacts between heparin and residues inthe N-domain occur. Surprisingly though, our analysis also identifies anumber of critical heparin contacts with four positively chargedresidues in the K1 domain. More surprisingly, we have furtherdemonstrated that heparin binding to these positively charged residuesin the K1 domain inhibits activity, and that mutagenesis of the residuesprovides NK1 variants with higher than wild-type activity. Such variantsare useful for the production of agonists for the promotion of cellgrowth, particularly for angiogenesis, and the treatment ofcardiovascular, hepatic, musculoskeletal and neuronal diseases.

[0015] Thus, the present invention provides a polypeptide variant of SEQID NO:1, said variant having the sequence of SEQ ID NO:1 apart from asubstitution or deletion of least one of positions corresponding to 132,134, 170 and 181 of HGF/SF. For ease of reference, positions of SEQ IDNO:1 are defined in relation to full length HGF/SF (SEQ ID NO:2) unlessstated to the contrary. The variant is one which retains the ability toexhibit heparin-dependent dimerization in solution and to act as anagonist against the MET receptor.

[0016] The invention also provides a polypeptide which is fragment ofthe polypeptide variant of the invention, said fragment retaining the132-181 region and further retaining the ability to exhibitheparin-dependent dimerization in solution and to act as an agonistagainst the MET receptor.

[0017] The invention further provides a composition comprising apolypeptide of the invention together with a pharmaceutically acceptablediluent or carrier.

[0018] The invention further provides a method for stimulating thegrowth of a cell which expresses the MET receptor, said methodcomprising bringing a polypeptide of the invention into contact withsaid cell. The cell may be in vitro or in vivo.

[0019] The invention further provides a method of treatment of a patienthaving a disease condition which requires stimulation of cell growth,said method comprising administering to a patient an effective amount ofa polypeptide of the invention.

[0020] The invention also provides a polypeptide of the invention foruse in a method of treatment of the human or animal body.

[0021] The invention further provides a polynucleotide coding for apolypeptide of the invention, as well as vectors carrying saidpolynucleotide, including expression vectors wherein the polynucleotideis operably linked to a promoter.

[0022] The invention further provides a host cell carrying a vector ofthe invention, and methods for the production of a polypeptide of theinvention which comprises culturing the host cells under conditionssuitable for the expression of the polynucleotide carried by the vector,and recovering the polypeptide from the cell or culture.

[0023] A major effort is underway for developing MET antagonists andagonists for therapy. MET antagonists are expected to find applicationsin a variety of epithelial tumours over-expressing MET, while receptoragonists may be valuable in liver regeneration, the repair of skinwounds and therapeutic angiogenesis. The structural and mutagenesis dataprovided by the present invention enables the generation of potent METagonists.

DETAILED DESCRIPTION OF THE INVENTION

[0024] Polypeptides

[0025] Polypeptides of the invention are those in which one of positions132, 134, 170 and 181 are substituted with any other amino acid. It ispreferred, however, that the substitutions are those which result in achange of charge. Preferred substitutions thus include reverse chargesubstitutions of aspartic acid and glutamic acid.

[0026] Two or more of the positions may be substituted simultaneously.Where two substitutions are made, in a preferred aspect the two areeither 132 and 134 or 170 and 181. Three substitutions may also be madeor all four positions may be substituted. Where more than one positionis substituted the substitutions may be the same or different.

[0027] Polypeptides of the invention may be prepared in isolated form.Isolated polypeptides of the invention will be those as defined above inisolated form, free or substantially free of material with which it isnaturally associated such as other polypeptides with which it is foundin the cell. The polypeptides may of course be formulated with diluentsor adjuvants and still for practical purposes be isolated. Thepolypeptides may be glycosylated, either naturally or by systems ofheterologous eukaryotic cells, or they may be (for example if producedby expression in a prokaryotic cell) unglycosylated.

[0028] A polypeptide of the invention may also be in a substantiallypurified form, in which case it will generally comprise the polypeptidein a preparation in which more than 90%, e.g. 95%, 98% or 99% of thepolypeptide in the preparation is a polypeptide of the invention.

[0029] Polypeptides of the invention may be modified for example by theaddition of histidine residues to assist their purification or by theaddition of a signal sequence to promote their secretion from a cell.

[0030] SEQ ID NO: 1 represents the wild-type human form of NK1 (togetherwith short N- and C-terminal extension). However, those of skill in theart will appreciate that in addition to the specific substitutions ofthe invention which result in increased activity, other positions of thewild-type molecule may be varied to a small degree without significantlyaffecting the overall function or structure of the polypeptide. Forexample, conservative substitutions can be made to many parts ofproteins with no discernable impact on the structure or function of thatprotein. Those of skill in the art will appreciate that a small number,for example from 1-20, e.g. 2, 3, 4 or 5-10 other amino acidsubstitutions mainly made to NK1 and provided substitutions do notsignificantly alter the activity of polypeptides of the invention suchvariants are still regarded as NK1 polypeptides.

[0031] Fragments of the polypeptide variants of the invention whichretain the region 132-181 also form a further part of the invention.Such fragments may be from 70 to 190 amino acids in size, for examplefrom 100 to 180 in size. An example of such a fragment is providedherein as the NK1 fragment of amino acids 32-206. Another fragment isone which includes at least 70 contiguous amino acids of the Kringle 1domain, which is found at 128-206. Preferably the fragment contains thisdomain in its entirety.

[0032] Methods to determine whether a polypeptide of the inventionretains the ability to exhibit heparin-dependent dimerization insolution are set out in the accompanying examples. As indicated, thepolypeptide may be incubated in with an equimolar concentration ofheparin in a 300 mM NaCl buffer and analysed by gel filtration orwestern blotting.

[0033] Likewise, the ability to act as an agonist of the MET receptormay be determined in accordance with the accompanying examples. This mayinvolve incubating the polypeptide with murine keratinocyte cells (e.g.MK cells) at a concentration of 10⁻¹⁰ M or higher (e.g. 10⁻¹⁰ to 10⁻⁸ M,and determining whether there is an increase in DNA synthesis in thecells.

[0034] A polypeptide according to the present invention may be isolatedand/or purified (e.g. using an antibody) for instance after productionby expression from encoding nucleic acid (for which see below).Polypeptides according to the present invention may also be generatedwholly or partly by chemical synthesis, for example in a step-wisemanner. The isolated and/or purified polypeptide may be used informulation of a composition, which may include at least one additionalcomponent, for example a pharmaceutical composition including apharmaceutically acceptable excipient, vehicle or carrier. A compositionincluding a polypeptide according to the invention may be used inprophylactic and/or therapeutic treatment as discussed below.

[0035] A polypeptide of the invention may be labelled with a revealinglabel. The revealing label may be any suitable label which allows thepolypeptide to be detected. Suitable labels include radioisotopes, e.g.¹²⁵I, enzymes, antibodies, polynucleotides and linkers such as biotin.Labelled polypeptides of the invention may be used in diagnosticprocedures such as immunoassays in order to determine the amount of apolypeptide of the invention in a sample.

[0036] Polypeptides and compositions thereof according to the inventionmay be used in methods of treatment. Such treatment will be directed topromoting the growth of cells in the human body which express the METreceptor. Such therapy will be useful for the promotion of angiogenesisand thus will be useful for the treatment of chronic skin wounds,chronic liver and kidney disease, degenerative musculoskeletelal andneuronal diseases and cardiovascular disease.

[0037] A particular use of the polypeptides of the invention is in thetreatment or prevention of liver damage caused by intoxication byN-acetyl-p-aminophenol (known commercially as paracetamol oracetaminophen). We have found that administration of a polypeptide ofthe invention in a mouse model substantially increases survival rates ofmice following administration of a lethal dose of paracetamol. Someprotection is also provided by the NK1 peptide itself. Thus in thisaspect of the invention, there is also provided the use of an NK1peptide for the above-mentioned treatment.

[0038] Thus the invention provides a method of treatment or preventionof liver damage in a subject who has ingested N-acetyl-p-aminophenol,the method comprising administering to the subject an effective amountof a polypeptide of the invention or an NK1 polypeptide.

[0039] The invention also provides a polypeptide of the invention or anNK1 polypeptide for use in a method of therapy of a human or animalsubject, particularly for treatment or prevention of liver damage in asubject who has ingested N-acetyl-p-aminophenol.

[0040] We have also demonstrated that the NK1 peptide as well as the 1K1polypeptide is effective in treating acute liver failure caused byα-amanitin, which is a potent specific inhibitor of RNA polymerase II.Thus the NK1 peptide as well as the peptides of the invention as definedherein may be used generally in a method of treatment of liver disease,particularly disease conditions associated with liver failure. Suchconditions include not only toxicity caused by N-acetyl-aminophenonl,but also include other drug-induced and other causes of liver failure,or disease.

[0041] Thus these peptides may be used to treat or prevent acute liverfailure or disease induced by toxins, including a toxin selected frommushroom poisoning (e.g. Amanita phalloides), arsenic, carbontetrachloride (or other chlorinated hydrocarbons), copper, ethanol,iron, methotrexate and phosphorus.

[0042] The invention may further be used to treat or prevent liverfailure or disease caused by other means, including conditions selectedfrom viral infection (such as by infection with a hepatitis virus, e.g.HAV, HBV or HCV), or other acute viral hepatitis, autoimmune chronichepatitis, acute fatty liver of pregnancy, Budd-Chiari syndrome andveno-occlusive disease, hyperthermia, hypoxia, malignant infiltration,Reye's syndrome, sepsis, Wilson's disease and in transplant rejection.

[0043] Polypeptides may be administered in any suitable form, forexample in a pharmaceutical composition such as water, saline, dextrose,glycerol, ethanol and the like. Compositions may be formulated forinjection, for example for direct injection to the site of intendedtreatment or intravenous injection.

[0044] Suitable doses of polypeptides will ultimately be at thediscretion of the physician taking account of the nature of thecondition to be treated and the condition of the patient. In general,dosage ranges will be 1 μg to 1 mg per kg body weight. The polypeptidesmay be administered by any suitable route, e.g. by i.v. or i.pinjection, or directly to the site of treatment.

[0045] By “treatment” it will be understood that this refers to anyadministration of a polypeptide intended to alleviate the severity of adisease being treated, to provide relief from the symptoms of thedisease or to prevent or slow down the development of the disease in anindividual with a disease condition or at risk of developing the diseasecondition.

[0046] Polynucleotides.

[0047] A polynucleotide of the invention is one which encodes apolypeptide of the invention as defined above. This includes DNA and RNApolynucleotides. A polynucleotide of the invention may be single ordouble stranded.

[0048] Generally, a polynucleotide according to the present invention isprovided as an isolate, in isolated and/or purified form, or free orsubstantially free of material with which it is naturally associated,such as free or substantially free of nucleic acid flanking the gene inthe human genome, except possibly one or more regulatory sequence(s) forexpression.

[0049] Sequences encoding all or part of the polypeptides of theinvention and/or its regulatory elements can be readily prepared by theskilled person using the information and references contained herein andtechniques known in the art (for example, see Sambrook, Fritsch andManiatis, “Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, 1989, and Ausubel et al, Short Protocols in MolecularBiology, John Wiley and Sons, 1992). These techniques include the use ofsite directed mutagenesis of nucleic acid encoding NK1, as described inthe accompanying examples.

[0050] Vectors.

[0051] Polynucleotides of the invention can be incorporated into arecombinant replicable vector. The vector may be used to replicate thenucleic acid in a compatible host cell. Thus in a further embodiment,the invention provides a method of making polynucleotides of theinvention by introducing a polynucleotide of the invention into areplicable vector, introducing the vector into a compatible host celland growing the host cell under conditions which bring about replicationof the vector. The vector may be recovered from the host cell. Suitablehost cells are described below in connection with expression vectors.

[0052] Expression Vectors.

[0053] Preferably, a polynucleotide of the invention in a vector isoperably linked to a control sequence which is capable of providing forthe expression of the coding sequence by the host cell., i.e. the vectoris an expression vector.

[0054] The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences.

[0055] Suitable vectors can be chosen or constructed, containingappropriate regulatory sequences, including promoter sequences,terminator fragments, polyadenylation sequences, enhancer sequences,marker genes and other sequences as appropriate. Vectors may beplasmids, viral e.g. ‘phage phagemid or baculoviral, cosmids, YACs,BACs, or PACs as appropriate. Vectors include gene therapy vectors, forexample vectors based on adenovirus, adeno-associated virus, retrovirus(such as HIV or MLV) or alpha virus vectors.

[0056] The vectors may be provided with an origin of replication,optionally a promoter for the expression of the said polynucleotide andoptionally a regulator of the promoter. The vectors may contain one ormore selectable marker genes, for example an ampicillin resistance genein the case of a bacterial plasmid or a neomycin resistance gene for amammalian vector. Vectors may be used in vitro, for example for theproduction of RNA or used to transfect or transform a host cell. Thevector may also be adapted to be used in vivo, for example in methods ofgene therapy. Systems for cloning and expression of a polypeptide in avariety of different host cells are well known. Suitable host cellsinclude bacteria, eukaryotic cells such as mammalian and yeast, andbaculovirus systems. Mammalian cell lines available in the art forexpression of a heterologous polypeptide include Chinese hamster ovarycells, HeLa cells, baby hamster kidney cells, COS cells and many others.

[0057] Promoters and other expression regulation signals may be selectedto be compatible with the host cell for which the expression vector isdesigned. For example, yeast promoters include S. cerevisiae GAL4 andADH promoters, S. pombe nmt1 and adh promoter. Mammalian promotersinclude the metallothionein promoter which is can be included inresponse to heavy metals such as cadmium. Viral promoters such as theSV40 large T antigen promoter or adenovirus promoters may also be used.All these promoters are readily available in the art.

[0058] The vectors may include other sequences such as promoters orenhancers to drive the expression of the inserted nucleic acid, nucleicacid sequences so that the polypeptide is produced as a fusion and/ornucleic acid encoding secretion signals so that the polypeptide producedin the host cell is secreted from the cell.

[0059] Vectors for production of polypeptides of the invention of foruse in gene therapy include vectors which carry a mini-gene sequence ofthe invention.

[0060] Vectors may be introduced into a suitable host cell as describedabove to provide for expression of a polypeptide of the invention. Thus,in a further aspect the invention provides a process for preparingpolypeptides according to the invention which comprises cultivating ahost cell carrying an expression vector as described above underconditions to provide for expression by the vector of a coding sequenceencoding the polypeptides, and recovering the expressed polypeptides.Polypeptides may also be expressed in in-vitro systems, such asreticulocyte lysate.

[0061] A further embodiment of the invention provides host cellscarrying the vectors for the replication and expression ofpolynucleotides of the invention. The cells will be chosen to becompatible with the said vector and may for example be bacterial, yeast,insect or mammalian.

[0062] The introduction of vectors into a host cell may be followed bycausing or allowing expression from the nucleic acid, e.g. by culturinghost cells (which may include cells actually transformed although morelikely the cells will be descendants of the transformed cells) underconditions for expression of the gene, so that the encoded polypeptideis produced. If the polypeptide is expressed coupled to an appropriatesignal leader peptide it may be secreted from the cell into the culturemedium. Following production by expression, a polypeptide may beisolated and/or purified from the host cell and/or culture medium, asthe case may be, and subsequently used as desired, e.g. in theformulation of a composition which may include one or more additionalcomponents, such as a pharmaceutical composition which includes one ormore pharmaceutically acceptable excipients, vehicles or carriers (e.g.see below).

[0063] A further aspect of the present invention provides a host cellcontaining nucleic acid as disclosed herein. The polynucleotides andvectors of the invention may be integrated into the genome (e.g.chromosome) of the host cell. Integration may be promoted by inclusionof sequences which promote recombination with the genome, in accordancewith standard techniques. The nucleic acid may be on anextra-chromosomal vector within the cell.

[0064] In the accompanying examples we show that wt-NK1 behaved as apartial agonist, as expected from the prior art. It produced fulldispersion of MDCK colonies and stimulation of DNA synthesis in MK cells(FIG. 1). Interestingly, maximal stimulation of DNA synthesis by wt-NK1occurred at concentrations as low as 10⁻¹⁰ M, a concentration much lowerthan those required in other studies see for example (Schwall et al.,1996). The higher potency of NK1 observed in our studies may reflect thesource (yeast vs. bacterial), and hence the activity, of the proteinused.

[0065] While wt-NK1 remains less active than full length HGF/SF,remarkably the two K domain mutants exhibited biological activity muchhigher than wt-NK1 and equal or higher to full length HGF/SF. Ourbiochemical data suggest that the K domain mutations result in increasednet affinity of NK1 for heparin. Thus the patch of amino acidsconsisting of K132, R134 and R181 acts as a negative effector of heparinbinding to NK1 and reverse-charge mutations of these residues increasesheparin binding probably via the main site in the N domain.

[0066] Regardless of the mechanism, we have demonstrated thatsubstitution of two amino acids in the K domain (K132:R134 or K170:R181)is sufficient for converting NK1 into a full receptor agonist. NK1, butnot HGF/SF, can be produced in yeast and is expected to exhibitfavourable in vivo kinetics and tissue distribution compared to fulllength HGF/SF.

Example 1

[0067] In this example the production and analysis of the biologicalactivity of two NK1 variants of the invention is illustrated. We showthat these variants undergo dimerization in a manner similar to NK1 inthe presence of heparin and are rendered potent receptor agoniststhrough mutagenesis of a cluster of positively-charged residues on the Kdomain.

[0068] Materials and Methods

[0069] Cloning, mutagenesis, expression and purification Cloning,expression and purification of wt-NK1 were carried out as described in(Chirgadze et al., 1999), except that final purification of NK1 bycation exchange chromatography was carried out on a Source15S column(Amersham Pharmacia Biotech). For expression of NK1 mutants, anEcOR1-NotI fragment from the wt-NK1 expression construct in pPIC9K wascloned into the pBluescript KS-vector (Stratagene) and DNA amplificationreactions were carried out using complementary pairs of mutagenicoligonucleotides. The N-domain mutants were produced by DNAamplification of the relevant fragments from full length human HGF/SFcDNA which carried R73E:R76E mutations (mutant HP11) or the(K58E:K60E:K62E) mutations (mutant HP12). N domain and K domain mutantswere finally cloned in the expression vector pPIC9K. Transformation andselection of P. pastoris was carried out as described previously(Chirgadze et al., 1999).

[0070] Purification of Heparin Fragments

[0071] Sodium heparin from bovine lung (Upjohn) was digested withheparinase I (Leo Pharmaceuticals) for 14 min at 37° C. in 10 mMphosphate buffer, 1.25 mM CaCl₂, pH 7.0. The water was evaporated, theresidue dissolved in 20 g/l ammonium bicarbonate and loaded onto aBiogel P-10 (Biorad) column. Fractions containing heparin fragments ofthe same length (up to hexadecasaccharide) were combined and water andammonium bicarbonate evaporated on a Rotavapor (Buechi). The heparinfragments were then dissolved in 0.1 M ammonium acetate and an aliquotwas run through a G3000 SW XL (30 cm×7.8 mm) and a G2000 SW XL (30cm×7.8 mm) GPC column on a HPLC system (Gilson) in order to assesspurity and concentration. The fragments were next lyophilized andredissolved in water (3 cycles) in order to eliminate ammonium acetate.

[0072] Characterization of wt- and Mutant NK1-Heparin Complexes

[0073] This was carried out by gel filtration chromatography andcross-linking experiments. For gel filtration, wt- or mutant NK1 (0.5mg/ml) were incubated for 2 hours in the presence or absence ofequimolar concentration of 14-mer heparin in phosphate buffered saline(PBS) adjusted to 300 mM NaCl. Samples were then loaded onto an HR30Superdex 200 column (Ammersham Pharmacia Biotech) and eluted at 0.5ml/min.

[0074] For cross-linking, 10 μl of wt- or mutant-NK1 (0.1 mg/ml) wereincubated in the absence or presence of an equimolar concentration of14-mer heparin in PBS. After 2 hours incubation at room temperature, 1μl of crosslinker (BS³, Pierce) was added at 100 fold molar excess, thereaction was continued for 30 minutes and then quenched with 1 μl of 1MTris-Cl, pH 7.4. Reaction products were loaded onto 15%SDS-polyacrylamide gels and blotted onto a nitrocellulose membrane(Schleicher & Schuell). The membrane was blocked in 2% skimmed milk,incubated for 1 hour in the presence of sheep anti-HGF/SF polyclonalantibody (1W53, 1:1000), washed with PBS+0.2% Tween 20 and nextincubated for 1 hour with HRP-conjugated anti-sheep immunoglobulinantibody (Dako). HRP activity was detected after 3 further washes inPBS+0.2% Tween 20 using a chemiluminescent substrate (Pierce).

[0075] MDCK Colony Scatter Assays

[0076] Scatter assays were carried out as described in (Gherardi et al.,1989; Stoker et al., 1987). Briefly, MDCK cells were plated at 1-2.5×10³cells/60 mm dish and cultured in 5% fetal calf serum in DMEM for 2-3days before addition of HGF/SF or wt- or mutant NK1. After overnightincubation, plates were inspected and several colonies from each platewere photographed using a Leica DM IRB inverted microscope equipped withphase contrast optics and a Hamamatsu colour chilled 3CCD camera.

[0077] DNA Synthesis in MK Cells

[0078] The mouse keratinocyte line MK was cultured to confluence inkeratinocyte SFM medium (Gibco) supplemented with 5 ng/ml EGF-53 and 50g/ml bovine pituitary extract (BPE) in 24 well tissue culture plates(Costar). At confluence, complete medium was replaced with basal medium(no EGF and BPE) for 24 h before addition of 1 Ci/well ³H-thymidine inbasal medium containing 1 mg/ml BSA and HGF/SF or NK1 proteins at theconcentrations specified in the legend to FIG. 1. After 16 hours thecells were transferred on ice, washed with PBS and incubated inice-cold, 5% trichloroacetic acid (TCA) for 30 min. TCA insolubleradioactivity was measured by scintillation after 2 washes with waterand lysis in 0.2 M NaOH for 30 minutes at 37C.

[0079] Results

[0080] The NK1 fragment of HGF/SF (amino acids 28-210) was expressed inthe methylotrophic yeast P. pastoris as described (Chirgadze et al.,1999) and crystallized in complex with a tetrahexameric (14-mer) heparinfragment. The heparin fragment was prepared by digestion andpurification from polydisperse heparin extracted from bovine lung.

[0081] The crystallization of the protein in complex with heparin isdescribed in GB0110430.6 of 27 Apr. 2001, from which priority is herebyclaimed and the contents of which are hereby incorporated by reference,and in Lietha, D. et al.; EMBO J. 2001 Oct. 15;20(20):5543-55, whosecontents are also herby incorporated by reference. The crystallizationand analysis of the complex allowed the present inventors to identifyamino acid residues in NK1 which could be altered. Having identifiedsuch residues, those of skill in the art to produce variants of theinvention based on the present disclosure herein.

[0082] Briefly, two crystal types were found. The asymmetric unit ofcrystal-type A contains two NK1 protomers, A and B, assembled into ahead-to-tail dimer, as in the previously described crystal structures ofNK1 (Chirgadze et al., 1999; Ultsch et al., 1998). A hepes molecule isbound to each of the K domains in the putative lysine-binding pocket, asin the lbht structure (Ultsch et al., 1998). A heparin molecule (H) wasclearly seen bound to the N domain of protomer A but the N domain ofprotomer B is partially disordered and poorly defined at the periphery.Thus it could not be seen whether a heparin molecule was bound. Theheparin molecule bound to protomer A also makes contacts with thekringle domain of protomer A′ from the neighbouring asymmetric unit ofthe crystals. The final refined structure contained 5 heparin sugarunits: 2 glycosamines (GlcN) and 3 iduronic acids (IdoA) of the 14present in the complex.

[0083] In contrast, the asymmetric unit of crystal type B contained anassembly of four NK1 dimers (A & B, C & D, E & F, G & H) with six boundheparin molecules. The dimers in the asymmetric unit were positioned ina circle with a pseudo-two fold axis running through the centre. The NK1dimer arrangement was identical to that observed in crystal type A anddescribed earlier (Chirgadze et al., 1999; Ultsch et al., 1998). Unlikethe structure of crystal type A, all residues between 38 and 208 arewell ordered and show clear electron density in all protomets. All Ndomains interact with heparin molecules. The N domains of protomers Aand E share a heparin molecule as do N domains of protomers D and G. Thelongest heparin fragment, that could be built into electron densitymaps, is nine sugar units in length; it is bound to the N domain ofprotomer C and the K domain of protomer F. Other heparin molecules areless defined with the shortest fragment containing only five sugar units(heparin molecule N). Each K domain has a hepes molecule bound in thesame binding pocket as in the structure of crystal type A. The dimerswithin the asymmetric unit show a good agreement with r.m.s.d. values ofCα atoms between 0.50 Å (comparing dimer consisting of protomers A and Bwith that consisting of protomers G and H) and 1.32 Å (comparing dimerconsisting of protomers A and B with that consisting of protomer C andD). The NK1 dimers in crystal type B are also very similar to the dimerin crystal type A, with the worst r.m.s.d. of Cα atoms amounting to 1.19Å for the dimer consisting of protomers A and B.

[0084] Heparin—K domain interactions were seen in both crystalstructures and involved a cluster of positively charged residues (K132,R134, K170, R181). These residues form a patch of positive electrostaticpotential lining against the negatively charged heparin chain. Thefunctional significance of the heparin—K domain interactions was probedby mutagenesis.

[0085] Novel NK1 Mutants

[0086] Two reverse-charge N domain mutants (HP11 and HP12) and two Kdomain mutants (1K1 and 1K2) were generated (Table 1) and characterizedfor heparin binding and biological activity. TABLE 1 NK1 MutantSubstitutions HP11 R73E: R76E HP12 K58E: K60E: K62E 1K1 K132E: R134E 1K2K170E: R181E

[0087] Cross-linking (Schwall et al., 1996) and gel filtration(Chirgadze et al., 1999) experiments were employed first in order tocharacterize heparin-mediated oligomerization of wt NK1 in solution.Wild-type and mutant NK1 were incubated in the absence or presence ofequimolar concentrations of 14-mer heparin. Cross-linked proteins wereanalyzed by western blotting and detected with an anti-HGF/SF polyclonalantibody (1W53). In addition, gel filtration of wild type and mutant NK1in the absence or presence of equimolar concentrations of 14-mer heparinwas also performed. Chromatography was carried out on an HR30Superdex-200 column equilibrated in PBS adjusted to 300 mm NaCl. Wt-NK1and the different mutants showed slight variations in elution volume dueto residual interaction with the column.

[0088] Heparin failed to induce both cross-linking and oligomerizationin solution of the HP11 mutant. HP12, the second N domain mutant wascross-linked by heparin but, like the HP11 mutant, failed to oligomerizein solution in the presence of heparin. The two K domain mutants (1K1and 1K2), however, behaved like wt-NK1 in these experiments. Inconclusion, the amino acids that make crystallographic contact withheparin in the N domain are required for heparin-dependent dimerizationof NK1 in solution indicating that these amino acids are responsible forheparan sulphate-dependent dimerization of NK1 on the cell surface.

[0089] Biological Activity of NK1 Mutants

[0090] Experiments with heparan sulphate-deficient cells haveestablished an essential requirement for heparan sulphate or solubleheparin for the biological activity of NK1 (Schwall et al., 1996).Normal cells display membrane-bound heparan sulphate and thus, if theyexpress the MET receptor, respond to NK1. They may fail, however, torespond to heparan-sulphate-deficient NK1 mutants such as HP11 and HP12.

[0091] Colony dispersion (scatter) assays with MDCK cells were performedessentially as described by Gherardi et al., 1989 and Stoker et al.,1987 in the presence of full length HGF/SF or NK1 proteins. Briefly,MDCK cells were plated at low density in 60 mm dishes and cultured for 3days in standard medium after which the medium was replaced with freshmedium or medium containing 10⁻¹⁰ M HGF/SF or 10⁻⁸ M of the various NK1proteins. After overnight incubation several colonies from each dishwere photographed using phase contrast optics.

[0092] The colonies in control cultures exhibited strong cell-celladhesion and a typical epithelial, ‘cobblestone’ appearance. HGF/SF(10⁻¹⁰ M), wt-NK1 or the K domain mutant 1K1 (10⁻⁸ M) induced fulldissociation of MDCK colonies. In contrast, both the HP11 and HP12mutants (10⁻⁸ M) were inactive. Addition of soluble heparin (10⁻⁶ M) didnot affect control cultures or cultures containing HGF/SF or NK1proteins.

[0093] The biological activity of the NK1 mutants was studied further ona different target, the MK mouse keratinocyte line, which exhibits astrong mitogenic response to HGF/SF (Moorby et al., 1995). Wt-NK1, butnot the N domain mutants HP11 and HP12, induced appreciable stimulationof DNA synthesis at concentrations of 10⁻¹⁰ M or higher (FIG. 1).Remarkably, the K domain mutant 1K1 exhibited activity much higher thanwt-NK1 and comparable or even higher than that of full length HGF/SF.1K2, the second K domain mutant, behaved like and gave a similar resultas the 1K1 mutant. Thus, the HP11 and HP12 mutations that failed toinduce dimerization of NK1 in solution, also caused loss of biologicalactivity, presumably due to the inability of these mutants to bindcell-associated heparan sulphate on the surface of MDCK or MK cells. Incontrast, the K domain mutations conferred increased biological activityto NK1 and converted it to a full receptor agonist.

[0094] In order to establish whether the loss of activity of the HP11and HP12 mutants was due to defective receptor binding or activation,competition experiments were carried out in which MDCK cells werecultured in the presence of HGF/SF alone (10⁻¹⁰ M) or in the presence ofHGF/SF and excess concentrations (10⁻⁸ or 10⁻⁷ M) of wt-NK1 or the two Ndomain mutants. As expected, wt-NK1 behaved as a partial antagonist butHP11 and HP12 exhibited no (HP11) or very little (HP12) antagonisticactivity implying that the lack of activity of these mutants is due toreduced receptor binding rather than failure to induce receptoractivation.

Example 2 In Vivo Activity of 1K1

[0095] Three groups of 12 Balb/c male mice (10 weeks old, about 35 g)plus a control group of 20 such animals were administered 0.6 g/kg i.p.of N-acetyl-p-aminophenol in 0.3 ml PBS. Following dosing, mice weretreated at two hours and six hours with 0.5 mg/kg i.v. of 1K1, NK1 orHGF/SF or, in the case of the control group, left untreated.

[0096] The results are shown in FIG. 2. Briefly, N-acetyl-p-aminophenolcaused death in 85% of the animals that received drug but no growthfactor over a period of 3 days, with just under 50% of the animals dead4 hours after treatment. HGF/SF offered some protection and, somewhatsurprisingly, NK1 was more active than HGF/SF, achieving a 40% survival3 days after treatment. The NK1 mutant 1K1 was the most effective of theprotein tested resulting in 80% survival.

Example 3 Activity of NK1 and 1K1 in -Amanitin-Induced Liver Failure

[0097] Three groups of 12 test mice and a control group of 20 mice ofthe same strain, size and sex as Example 2 were administered by i.p. 0.9mg/kg α-amanitin. The test groups were then given 5 injections every 12hours, commencing 12 hours after α-amanitin dosing, of 0.5 mg/kg i.v. ofNK1, 1K1 or HGF/SF. The results are shown in FIG. 3, indicating thatboth 1K1 and NK1 were effective in reducing early stage (3-5 days)hepatic toxicity.

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1 2 1 183 PRT Homo sapiens 1 Tyr Ala Glu Gly Gln Arg Lys Arg Arg Asn ThrIle His Glu Phe Lys 1 5 10 15 Lys Ser Ala Lys Thr Thr Leu Ile Lys IleAsp Pro Ala Leu Lys Ile 20 25 30 Lys Thr Lys Lys Val Asn Thr Ala Asp GlnCys Ala Asn Arg Cys Thr 35 40 45 Arg Asn Lys Gly Leu Pro Phe Thr Cys LysAla Phe Val Phe Asp Lys 50 55 60 Ala Arg Lys Gln Cys Leu Trp Phe Pro PheAsn Ser Met Ser Ser Gly 65 70 75 80 Val Lys Lys Glu Phe Gly His Glu PheAsp Leu Tyr Glu Asn Lys Asp 85 90 95 Tyr Ile Arg Asn Cys Ile Ile Gly LysGly Arg Ser Tyr Lys Gly Thr 100 105 110 Val Ser Ile Thr Lys Ser Gly IleLys Cys Gln Pro Trp Ser Ser Met 115 120 125 Ile Pro His Glu His Ser PheLeu Pro Ser Ser Tyr Arg Gly Lys Asp 130 135 140 Leu Gln Glu Asn Tyr CysArg Asn Pro Arg Gly Glu Glu Gly Gly Pro 145 150 155 160 Trp Cys Phe ThrSer Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile 165 170 175 Pro Gln CysSer Glu Val Glu 180 2 728 PRT Homo sapiens 2 Met Trp Val Thr Lys Leu LeuPro Ala Leu Leu Leu Gln His Val Leu 1 5 10 15 Leu His Leu Leu Leu LeuPro Ile Ala Ile Pro Tyr Ala Glu Gly Gln 20 25 30 Arg Lys Arg Arg Asn ThrIle His Glu Phe Lys Lys Ser Ala Lys Thr 35 40 45 Thr Leu Ile Lys Ile AspPro Ala Leu Lys Ile Lys Thr Lys Lys Val 50 55 60 Asn Thr Ala Asp Gln CysAla Asn Arg Cys Thr Arg Asn Lys Gly Leu 65 70 75 80 Pro Phe Thr Cys LysAla Phe Val Phe Asp Lys Ala Arg Lys Gln Cys 85 90 95 Leu Trp Phe Pro PheAsn Ser Met Ser Ser Gly Val Lys Lys Glu Phe 100 105 110 Gly His Glu PheAsp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys 115 120 125 Ile Ile GlyLys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys 130 135 140 Ser GlyIle Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His 145 150 155 160Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr 165 170175 Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser 180185 190 Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu195 200 205 Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu MetAsp 210 215 220 His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp His GlnThr Pro 225 230 235 240 His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro AspLys Gly Phe Asp 245 250 255 Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln ProArg Pro Trp Cys Tyr 260 265 270 Thr Leu Asp Pro His Thr Arg Trp Glu TyrCys Ala Ile Lys Thr Cys 275 280 285 Ala Asp Asn Thr Met Asn Asp Thr AspVal Pro Leu Glu Thr Thr Glu 290 295 300 Cys Ile Gln Gly Gln Gly Glu GlyTyr Arg Gly Thr Val Asn Thr Ile 305 310 315 320 Trp Asn Gly Ile Pro CysGln Arg Trp Asp Ser Gln Tyr Pro His Glu 325 330 335 His Asp Met Thr ProGlu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn 340 345 350 Tyr Cys Arg AsnPro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr 355 360 365 Asp Pro AsnIle Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp 370 375 380 Met SerHis Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met 385 390 395 400Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asp 405 410415 Lys Asn Met Glu Asp Leu His Arg His Ile Phe Trp Glu Pro Asp Ala 420425 430 Ser Lys Leu Asn Glu Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala His435 440 445 Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile Pro Trp Asp TyrCys 450 455 460 Pro Ile Ser Arg Cys Glu Gly Asp Thr Thr Pro Thr Ile ValAsn Leu 465 470 475 480 Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys GlnLeu Arg Val Val 485 490 495 Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly TrpMet Val Ser Leu Arg 500 505 510 Tyr Arg Asn Lys His Ile Cys Gly Gly SerLeu Ile Lys Glu Ser Trp 515 520 525 Val Leu Thr Ala Arg Gln Cys Phe ProSer Arg Asp Leu Lys Asp Tyr 530 535 540 Glu Ala Trp Leu Gly Ile His AspVal His Gly Arg Gly Asp Glu Lys 545 550 555 560 Cys Lys Gln Val Leu AsnVal Ser Gln Leu Val Tyr Gly Pro Glu Gly 565 570 575 Ser Asp Leu Val LeuMet Lys Leu Ala Arg Pro Ala Val Leu Asp Asp 580 585 590 Phe Val Ser ThrIle Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu 595 600 605 Lys Thr SerCys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn 610 615 620 Tyr AspGly Leu Leu Arg Val Ala His Leu Tyr Ile Met Gly Asn Glu 625 630 635 640Lys Cys Ser Gln His His Arg Gly Lys Val Thr Leu Asn Glu Ser Glu 645 650655 Ile Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly Asp 660665 670 Tyr Gly Gly Pro Leu Val Cys Glu Gln His Lys Met Arg Met Val Leu675 680 685 Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile Pro Asn Arg ProGly 690 695 700 Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp Ile His LysIle Ile 705 710 715 720 Leu Thr Tyr Lys Val Pro Gln Ser 725

1-18. (Canceled)
 19. A polypeptide comprising amino acids of SEQ IDNO:1, wherein the amino acid of at least one of positions 132, 134, 170,or 181 is substituted.
 20. The polypeptide of claim 19 wherein aminoacids at positions 132 and 134 are substituted.
 21. The polypeptide ofclaim 20, wherein said substitutions are K132E and R134E.
 22. Thepolypeptide of claim 19 wherein the amino acids at positions 170 and 181are substituted.
 23. The polypeptide of claim 22 wherein saidsubstitutions are K170E and R181E.
 24. A polypeptide comprising aminoacids 132-181 of SEQ ID NO:2, wherein said polypeptide exhibitsheparin-dependent dimerization and is a MET receptor agonist and whereinthe amino acid of at least one of positions 132, 134, 170 or 181 issubstituted.
 25. A composition comprising the polypeptide of any one ofclaims 19-24, and a pharmaceutically acceptable diluent or carrier. 26.A method for stimulating the growth of a cell expressing a MET receptor,said method comprising bringing the polypeptide of any one of claims19-24 into contact with said cell.
 27. A method of treating a patient inneed of stimulation of cell growth, said method comprising administeringto said patient an effective amount of the polypeptide of any one ofclaims 19-24.
 28. A method of treating or preventing liver damage in asubject who has ingested N-acetyl-p-aminophenol, the method comprisingadministering to the subject an effective amount of a polypeptideselected from an NK1 polypeptide or the polypeptide of any one of claims19-24.
 29. A method of treating or preventing liver failure, the methodcomprising administering to a subject in need of such treatment aneffective amount of a polypeptide selected from an NK1 polypeptide orthe polypeptide of any one of claims 19-24.
 30. A polynucleotide codingfor the polypeptide of any one of claims 19-24.
 31. An expression vectorcomprising the polynucleotide of claim 30 operably linked to a promoter.32. A host cell carrying the vector of claim 31.